The support function of Core C, Expression and Production, are three fold: 1) to express novel bioengineered antibody constructs in transient and stable mammalian expression systems, 2) to produce and purify these antibody constructs for preclinical studies planned in Project 2, Bioengineered Antibodies, and Project 3, Antibody Chelates and Conjugates, and 3) to produce and purify clinical grade material for the human imaging and Phase I/II therapy trials proposed in this Project. Novel anti-CEA bioengineered antibodies will be transiently expressed in a COS-1 cell based system. Transient expression will allow the rapid production and evaluation of novel bioengineered constructs for their proper folding, molecular size, affinity and avidity. If initial biochemical analysis looks promising, candidate constructs will be expressed in the Celltech glutamine synthetase (GS) mammalian expression system. Currently, expression of the cT84.66 GS18 Flex-ser minibody in the GS system has shown excellent results with high producing cell lines expressing 10-50 ug/m1 in stationary flasks. Cell culture supernatant for preclinical studies will be purified by ion exchange chromatography and an anti-idiotype affinity column. Antibody constructs will be produced in stationary T-flasks to provide 1 mg lots of purified material for in vitro characterization and animal pharmacokinetics studies. A limited number of site specific conjugation constructs, which show the desired criteria for further protein conjugation studies, will be produced and purified in 100 mg lots in a minifermenter bioreactor. Core C will select high-producing cell lines for three bioengineered antibody constructs: the cT84.66 minibody, the T84.66 diabody and a humanized T84.66 antibody. Bioengineered antibodies will be produced in a state-of-the-art hollow-fiber bioreactor. In addition to the novel bioengineered constructs, Core C will produce 4-5 grams of chimeric T8.66 antibody and generate F(ab')2 fragments for continuing human therapy trials. Antibody will be purified under protocols established in the previous Project period. The cell lines and purified antibody will meet the specifications set forth in the current FDA guidelines.